The impacts of diel thermal variability on growth, development and performance of wild Atlantic salmon (Salmo salar) from two thermally distinct rivers.

Temperature in many natural aquatic environments follows a diel cycle, but to date, we know little on how diel thermal cycles affect fish biology. The current study investigates the growth, development and physiological performance of wild Atlantic salmon collected from the Miramichi and Restigouche rivers (NB, Canada). Fish were collected as parr and acclimated to either 16-21 or 19-24°C diel thermal cycles throughout the parr and smolt life stages. Both Miramichi and Restigouche Atlantic salmon parr grew at similar rates during 16-21 or 19-24°C acclimations. However, as smolts, the growth rates of the Miramichi (-8% body mass day-1) and Restigouche (-38% body mass day-1) fish were significantly slower at 19-24°C, and were in fact negative, indicating loss of mass in this group. Acclimation to 19-24°C also increased Atlantic salmon CTmax. Our findings suggest that both life stage and river origin impact Atlantic salmon growth and performance in the thermal range used herein. These findings provide evidence for local adaptation of Atlantic salmon, increased vulnerability to warming temperatures, and highlight the differential impacts of these ecologically relevant diel thermal cycles on the juvenile life stages in this species.

Taurine depletion impairs cardiac function and affects tolerance to hypoxia and high temperatures in brook char (Salvelinus fontinalis).

Physiological and environmental stressors can cause osmotic stress in fish hearts, leading to a reduction in intracellular taurine concentration. Taurine is a ß-amino acid known to regulate cardiac function in other animal models but its role in fish has not been well characterized. We generated a model of cardiac taurine deficiency (TD) by feeding brook char (Salvelinus fontinalis) a diet enriched in ß-alanine, which inhibits cardiomyocyte taurine uptake. Cardiac taurine levels were reduced by 21% and stress-induced changes in normal taurine handling were observed in TD brook char. Responses to exhaustive exercise and acute thermal and hypoxia tolerance were then assessed using a combination of in vivo, in vitro and biochemical approaches. Critical thermal maximum was higher in TD brook char despite significant reductions in maximum heart rate. In vivo, TD brook char exhibited a lower resting heart rate, blunted hypoxic bradycardia and a severe reduction in time to loss of equilibrium under hypoxia. In vitro function was similar between control and TD hearts under oxygenated conditions, but stroke volume and cardiac output were severely compromised in TD hearts under severe hypoxia. Aspects of mitochondrial structure and function were also impacted in TD permeabilized cardiomyocytes, but overall effects were modest. High levels of intracellular taurine are required to achieve maximum cardiac function in brook char and cardiac taurine efflux may be necessary to support heart function under stress. Taurine appears to play a vital, previously unrecognized role in supporting cardiovascular function and stress tolerance in fish.

The physiological ups and downs of thermal variability in temperate freshwater ecosystems.

Freshwater fish face a variety of spatiotemporal thermal challenges throughout their life. On a broad scale, temperature is an important driver of physiological, behavioural and ecological patterns and ultimately affects populations and overall distribution. These broad patterns are partly underpinned by the small-scale local effects of temperature on individuals within the population. Climate change is increasing the range of daily thermal variation in most freshwater ecosystems, altering behaviour and performance of resident fishes. The aim of this review is understanding how daily thermal variation in temperate rivers affects individual fish physiology, behaviour and overall performance. The following are highlighted in this study: (a) the physical characteristics of rivers that can either buffer or exacerbate thermal variability, (b) the effects of thermal variability on growth and metabolism, (c) the approaches for quantifying thermal variation and thermal stress and (d) how fish may acclimatize or adapt to our changing climate.

Thermal variation near the thermal optimum does not affect the growth, metabolism or swimming performance in wild Atlantic salmon Salmo salar.

Typically, laboratory studies on the physiological effects of temperature are conducted using stable acclimation temperatures. Nonetheless, information extrapolated from these studies may not accurately represent wild populations living in thermally variable environments. The aim of this study was to compare the growth rate, metabolism and swimming performance of wild Atlantic salmon exposed to cycling temperatures, 16-21°C, and stable acclimation temperatures, 16, 18.5, 21°C. Growth rate, metabolic rate, swimming performance and anaerobic metabolites did not change among acclimation groups, suggesting that within Atlantic salmon's thermal optimum range, temperature variation has no effect on these physiological properties.

The endemic and endangered Maugean Skate (Zearaja maugeana) exhibits short-term severe hypoxia tolerance.

The endangered and range-restricted Maugean skate (Zearaja maugeana) is subjected to large environmental variability coupled with anthropogenic stressors in its endemic habitat, Macquarie Harbour, Tasmania. However, little is known about the basic biology/physiology of this skate, or how it may respond to future environmental challenges predicted from climate change and/or increases in human activities such as aquaculture. These skate live at a preferred depth of 5-15 m where the dissolved oxygen (DO) levels are moderate (~55% air saturation), but can be found in areas of the Harbour where DO can range from 100% saturation to anoxia. Given that the water at their preferred depth is already hypoxic, we sought to investigate their response to further decreases in DO that may arise from potential increases in anthropogenic stress. We measured oxygen consumption, haematological parameters, tissue-enzyme capacity and heat shock protein (HSP) levels in skate exposed to 55% dissolved O2 saturation (control) and 20% dissolved O2 saturation (hypoxic) for 48 h. We conclude that the Maugean skate appears to be an oxyconformer, with a decrease in the rate of O2 consumption with increasing hypoxia. Increases in blood glucose and lactate at 20% O2 suggest that skate are relying more on anaerobic metabolism to tolerate periods of very low oxygen. Despite these metabolic shifts, there was no difference in HSP70 levels between groups, suggesting this short-term exposure did not elicit a cellular stress response. The metabolic state of the skate suggests that low oxygen stress for longer periods of time (i.e. >48 h) may not be tolerable and could potentially result in loss of habitat or shifts in their preferred habitat. Given its endemic distribution and limited life-history information, it will be critical to understand its tolerance to environmental challenges to create robust conservation strategies.

The importance of incorporating natural thermal variation when evaluating physiological performance in wild species.

Environmental variability in aquatic ecosystems makes the study of ectotherms complex and challenging. Physiologists have historically overcome this hurdle in the laboratory by using 'average' conditions, representative of the natural environment for any given animal. Temperature, in particular, has widespread impact on the physiology of animals, and it is becoming increasingly important to understand these effects as we face future climate challenges. The majority of research to date has focused on the expected global average increase in temperature; however, increases in climate variability are predicted to affect animals as much or more than climate warming. Physiological responses associated with the acclimation to a new stable temperature are distinct from those in thermally variable environments. Our goal is to highlight these physiological differences as they relate to both thermal acclimation and the 'fallacy of the average' or Jensen's inequality using theoretical models and novel empirical data. We encourage the use of more realistic thermal environments in experimental design to advance our understanding of these physiological responses such that we can better predict how aquatic animals will respond to future changes in our climate.

Pass the salt: physiological consequences of ecologically relevant hyposmotic exposure in juvenile gummy sharks (Mustelus antarcticus) and school sharks (Galeorhinus galeus).

Estuarine habitats are frequently used as nurseries by elasmobranch species for their protection and abundant resources; however, global climate change is increasing the frequency and severity of environmental challenges in these estuaries that may negatively affect elasmobranch physiology. Hyposmotic events are particularly challenging for marine sharks that osmoconform, and species-specific tolerances are not well known. Therefore, we sought to determine the effects of an acute (48 h) ecologically relevant hyposmotic event (25.8 ppt) on the physiology of two juvenile shark species, namely the school shark (Galeorhinus galeus), listed by the Australian Environmental Protection and Biodiversity Conservation Act as 'conservation dependent', and the gummy shark (Mustelus antarcticus), from the Pittwater Estuary (Australia). In both species, we observed a decrease in plasma osmolality brought about by selective losses of NaCl, urea and trimethylamine N-oxide, as well as decreases in haemoglobin, haematocrit and routine oxygen consumption. Heat-shock protein levels varied between species during the exposure, but we found no evidence of protein damage in any of the tissues tested. Although both species seemed to be able to cope with this level of osmotic challenge, overall the school sharks exhibited higher gill Na+/K+-ATPase activity and ubiquitin concentrations in routine and experimental conditions, a larger heat-shock protein response and a smaller decrease in routine oxygen consumption during the hyposmotic exposure, suggesting that there are species-specific responses that could potentially affect their ability to withstand longer or more severe changes in salinity. Emerging evidence from acoustic monitoring of sharks has indicated variability in the species found in the Pittwater Estuary during hyposmotic events, and together, our data may help to predict species abundance and distribution in the face of future global climate change.

Physiological responses to hypersalinity correspond to nursery ground usage in two inshore shark species (Mustelus antarcticus and Galeorhinus galeus).

Shark nurseries are susceptible to environmental fluctuations in salinity because of their shallow, coastal nature; however, the physiological impacts on resident elasmobranchs are largely unknown. Gummy sharks (Mustelus antarcticus) and school sharks (Galeorhinus galeus) use the same Tasmanian estuary as a nursery ground; however, each species has distinct distribution patterns that are coincident with changes in local environmental conditions, such as increases in salinity. We hypothesized that these differences were directly related to differential physiological tolerances to high salinity. To test this hypothesis, we exposed wild, juvenile school and gummy sharks to an environmentally relevant hypersaline (120% SW) event for 48 h. Metabolic rate decreased 20-35% in both species, and gill Na(+)/K(+)-ATPase activity was maintained in gummy sharks but decreased 37% in school sharks. We measured plasma ions (Na(+), K(+), Cl(-)) and osmolytes [urea and trimethylamine oxide (TMAO)], and observed a 33% increase in plasma Na(+) in gummy sharks with hyperosmotic exposure, while school sharks displayed a typical ureosmotic increase in plasma urea (∼20%). With elevated salinity, gill TMAO concentration increased by 42% in school sharks and by 30% in gummy sharks. Indicators of cellular stress (heat shock proteins HSP70, 90 and 110, and ubiquitin) significantly increased in gill and white muscle in both a species- and a tissue-specific manner. Overall, gummy sharks exhibited greater osmotic perturbation and ionic dysregulation and a larger cellular stress response compared with school sharks. Our findings provide physiological correlates to the observed distribution and movement of these shark species in their critical nursery grounds.

Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism.

BACKGROUND: Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of ß-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms. RESULTS: Herein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARß/δ- and PPARα-dependent mechanism. Enhanced PPARß/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα(-/-) mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation. CONCLUSIONS: Nitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.

Oxidative stress and metabolic responses to copper in freshwater- and seawater-acclimated killifish, Fundulus heteroclitus.

In freshwater (FW), many of the main mechanisms of copper (Cu) toxicity have been characterized; however, toxicity mechanisms in seawater (SW) are less well understood. We investigated the effects of salinity on Cu-induced oxidative stress and metabolic responses in adult killifish, Fundulus heteroclitus. We exposed FW and SW-acclimated killifish to either low Cu (LC, 50 µg/L) or high Cu (HC, 200 µg/L) for 96 h and compared them to controls (CTRL) under the same salinities without added Cu. Cu exerted minimal influence on tissue ion levels in either FW or SW. Salinity generally protected against Cu bioaccumulation in the gills and liver, but not in the carcass. Hematocrit (Hct) and hemoglobin (Hb) levels were increased by LC and HC in both FW and SW, and blood lactate was reduced in FW-killifish exposed to LC and HC. Rates of oxygen consumption were similar across treatments. Salinity reduced Cu load in gill, liver and intestine at LC but only in the gills at HC. In general, Cu increased gill, liver, and intestine catalase (CAT) activity, while superoxide dismutase (SOD) either decreased or remained unchanged depending on tissue-type. These changes did not directly correlate with levels of protein carbonyls, used as an index of oxidative stress. Cu-induced changes in carbohydrate metabolic enzymes were low across tissues and the effect of salinity was variable. Thus, while salinity clearly protects against Cu bioaccumulation in some tissues, it is unclear whether salinity protects against Cu-induced oxidative stress and metabolic responses.

Muscle metabolic remodeling in response to endurance exercise in salmonids.

Phenotypic plasticity of skeletal muscle is relevant to swimming performance and metabolism in fishes, especially those that undergo extreme locomotory feats, such as seasonal migration. However, the influence of endurance exercise and the molecular mechanisms coordinating this remodeling are not well understood. The present study examines muscle metabolic remodeling associated with endurance exercise in fed rainbow trout as compared to migrating salmon. Trout were swum for 4 weeks at 1.5 BL/s, a speed similar to that of migrating salmon and red and white muscles were sampled after each week. We quantified changes in key enzymes in aerobic and carbohydrate metabolism [citrate synthase (CS), ß-hydroxyacyl-CoA dehydrogenase (HOAD), hexokinase (HK)] and changes in mRNA expression of major regulators of metabolic phenotype (AMPK, PPARs) and lipid (carnitine palmitoyltransferase, CPT I), protein (aspartate aminotransferase, AST) and carbohydrate (HK) oxidation pathways. After 1 week of swimming substantial increases were seen in AMPK and PPARα mRNA expression and of their downstream target genes, CPTI and HK in red muscle. However, significant changes in CS and HK activity occurred only after 4 weeks. In contrast, there were few changes in mRNA expression and enzyme activities in white muscle over the 4-weeks. Red muscle results mimic those found in migrating salmon suggesting a strong influence of exercise on red muscle phenotype. In white muscle, only changes in AMPK and PPAR expression were similar to that seen with migrating salmon. However, in contrast to exercise alone, in natural migration HK decreased while AST increased suggesting that white muscle plays a role in supplying fuel and intermediates possibly through tissue breakdown during prolonged fasting. Dissecting individual and potentially synergistic effects of multiple stressors will enable us to determine major drivers of the metabolic phenotype and their impacts on whole animal performance.

A product of its environment: the epaulette shark (Hemiscyllium ocellatum) exhibits physiological tolerance to elevated environmental CO2.

Ocean acidification, resulting from increasing anthropogenic CO2 emissions, is predicted to affect the physiological performance of many marine species. Recent studies have shown substantial reductions in aerobic performance in some teleost fish species, but no change or even enhanced performance in others. Notably lacking, however, are studies on the effects of near-future CO2 conditions on larger meso and apex predators, such as elasmobranchs. The epaulette shark (Hemiscyllium ocellatum) lives on shallow coral reef flats and in lagoons, where it may frequently encounter short-term periods of environmental hypoxia and elevated CO2, especially during nocturnal low tides. Indeed, H. ocellatum is remarkably tolerant to short periods (hours) of hypoxia, and possibly hypercapnia, but nothing is known about its response to prolonged exposure. We exposed H. ocellatum individuals to control (390 µatm) or one of two near-future CO2 treatments (600 or 880 µatm) for a minimum of 60 days and then measured key aspects of their respiratory physiology, namely the resting oxygen consumption rate, which is used to estimate resting metabolic rate, and critical oxygen tension, a proxy for hypoxia sensitivity. Neither of these respiratory attributes was affected by the long-term exposure to elevated CO2. Furthermore, there was no change in citrate synthase activity, a cellular indicator of aerobic energy production. Plasma bicarbonate concentrations were significantly elevated in sharks exposed to 600 and 880 µatm CO2 treatments, indicating that acidosis was probably prevented by regulatory changes in acid-base relevant ions. Epaulette sharks may therefore possess adaptations that confer tolerance to CO2 levels projected to occur in the ocean by the end of this century. It remains uncertain whether other elasmobranchs, especially pelagic species that do not experience such diurnal fluctuations in their environment, will be equally tolerant.

Genomic and metabolic preparation of muscle in sockeye salmon Oncorhynchus nerka for spawning migration.

Prolonged endurance exercise and fasting are two major metabolic challenges facing Pacific salmon during spawning migrations that often occur over 1,000 km. Because both prolonged exercise and fasting stimulate the oxidation of lipids, particularly in heavily recruited tissues such as muscle, we sought to investigate the regulatory mechanisms that establish and maintain the capacity for substrate oxidation at four separate locations during the final 750 km of nonfeeding migration in sockeye salmon Oncorhynchus nerka. Transcript levels of multiple genes encoding for important regulators of lipid, carbohydrate, and protein oxidation as well as the activity of several important enzymes involved in lipid and carbohydrate oxidation were examined in red and white muscle. We found in both muscle types that the messenger RNA (mRNA) expression of carnitine palmitoyltransferase I isoforms, peroxisome proliferator-activated receptors α and ß, and adenosine monophosphate-activated protein kinase ß1 were all significantly higher at the onset compared to later stages of nonfeeding migration. However, the activities of ß-hydroxyacyl-CoA dehydrogenase and citrate synthase were higher only early in migration and only in red muscle. Later in the migration and as muscle lipid stores were greatly depleted, the mRNA levels of hexokinase I and aspartate aminotransferase increased in white muscle. Overall, at the onset of migration, high transcript and metabolic enzyme activity levels in skeletal muscle of sockeye salmon may help support the high rates of lipid oxidation needed for endurance swimming. Furthermore, we suggest that the muscle capacity to use carbohydrates and proteins may be adjusted throughout migration on an as-needed basis to fuel burst exercise through very difficult hydraulic passages in the river and perhaps during mating activities.

Tissue-specific changes in fatty acid oxidation in hypoxic heart and skeletal muscle.

Exposure to hypobaric hypoxia is sufficient to decrease cardiac PCr/ATP and alters skeletal muscle energetics in humans. Cellular mechanisms underlying the different metabolic responses of these tissues and the time-dependent nature of these changes are currently unknown, but altered substrate utilization and mitochondrial function may be a contributory factor. We therefore sought to investigate the effects of acute (1 day) and more sustained (7 days) hypoxia (13% O2) on the transcription factor peroxisome proliferator-activated receptor α (PPARα) and its targets in mouse cardiac and skeletal muscle. In the heart, PPARα expression was 40% higher than in normoxia after 1 and 7 days of hypoxia. Activities of carnitine palmitoyltransferase (CPT) I and ß-hydroxyacyl-CoA dehydrogenase (HOAD) were 75% and 35% lower, respectively, after 1 day of hypoxia, returning to normoxic levels after 7 days. Oxidative phosphorylation respiration rates using palmitoyl-carnitine followed a similar pattern, while respiration using pyruvate decreased. In skeletal muscle, PPARα expression and CPT I activity were 20% and 65% lower, respectively, after 1 day of hypoxia, remaining at this level after 7 days with no change in HOAD activity. Oxidative phosphorylation respiration rates using palmitoyl-carnitine were lower in skeletal muscle throughout hypoxia, while respiration using pyruvate remained unchanged. The rate of CO2 production from palmitate oxidation was significantly lower in both tissues throughout hypoxia. Thus cardiac muscle may remain reliant on fatty acids during sustained hypoxia, while skeletal muscle decreases fatty acid oxidation and maintains pyruvate oxidation.

Acclimatization of skeletal muscle mitochondria to high-altitude hypoxia during an ascent of Everest.

Ascent to high altitude is associated with a fall in the partial pressure of inspired oxygen (hypobaric hypoxia). For oxidative tissues such as skeletal muscle, resultant cellular hypoxia necessitates acclimatization to optimize energy metabolism and restrict oxidative stress, with changes in gene and protein expression that alter mitochondrial function. It is known that lowlanders returning from high altitude have decreased muscle mitochondrial densities, yet the underlying transcriptional mechanisms and time course are poorly understood. To explore these, we measured gene and protein expression plus ultrastructure in muscle biopsies of lowlanders at sea level and following exposure to hypobaric hypoxia. Subacute exposure (19 d after initiating ascent to Everest base camp, 5300 m) was not associated with mitochondrial loss. After 66 d at altitude and ascent beyond 6400 m, mitochondrial densities fell by 21%, with loss of 73% of subsarcolemmal mitochondria. Correspondingly, levels of the transcriptional coactivator PGC-1α fell by 35%, suggesting down-regulation of mitochondrial biogenesis. Sustained hypoxia also decreased expression of electron transport chain complexes I and IV and UCP3 levels. We suggest that during subacute hypoxia, mitochondria might be protected from oxidative stress. However, following sustained exposure, mitochondrial biogenesis is deactivated and uncoupling down-regulated, perhaps to improve the efficiency of ATP production.

Regulation of carnitine palmitoyltransferase (CPT) I during fasting in rainbow trout (Oncorhynchus mykiss) promotes increased mitochondrial fatty acid oxidation.

Periods of fasting, in most animals, are fueled principally by fatty acids, and changes in the regulation of fatty acid oxidation must exist to meet this change in metabolic substrate use. We examined the regulation of carnitine palmitoyltransferase (CPT) I, to help explain changes in mitochondrial fatty acid oxidation with fasting. After fasting rainbow trout (Oncorhynchus mykiss) for 5 wk, the mitochondria were isolated from red muscle and liver to determine (1) mitochondrial fatty acid oxidation rate, (2) CPT I activity and the concentration of malonyl-CoA needed to inhibit this activity by 50% (IC(50)), (3) mitochondrial membrane fluidity, and (4) CPT I (all five known isoforms) and peroxisome proliferator-activated receptor (PPARα and PPARß) mRNA levels. Fatty acid oxidation in isolated mitochondria increased during fasting by 2.5- and 1.75-fold in liver and red muscle, respectively. Fasting also decreased sensitivity of CPT I to malonyl-CoA (increased IC(50)), by two and eight times in red muscle and liver, respectively, suggesting it facilitates the rate of fatty acid oxidation. In the liver, there was also a significant increase CPT I activity per milligram mitochondrial protein and in whole-tissue PPARα and PPARß mRNA levels. However, there were no changes in mitochondrial membrane fluidity in either tissue, indicating that the decrease in CPT I sensitivity to malonyl-CoA is not due to bulk fluidity changes in the membrane. However, there were significant differences in CPT I mRNA levels during fasting. Overall, these data indicate some important changes in the regulation of CPT I that promote the increased mitochondrial fatty acid oxidation that occurs during fasting in trout.

Changes in HIF-1α protein, pyruvate dehydrogenase phosphorylation, and activity with exercise in acute and chronic hypoxia.

Exercise under acute hypoxia elicits a large increase in blood lactate concentration ([La](b)) compared with normoxic exercise. However, several studies in humans show that with the transition to chronic hypoxia, exercise [La](b) returns to normoxic levels. Although extensively examined over the last decades, the muscle-specific mechanisms responsible for this phenomenon remain unknown. To assess the changes in skeletal muscle associated with a transition from acute to chronic hypoxia, CD-1 mice were exposed for 24 h (24H), 1 wk (1WH), or 4 wk (4WH) to hypobaric hypoxia (equivalent to 4,300 m), exercised under 12% O(2), and compared with normoxic mice (N) at 21% O(2). Since the enzyme pyruvate dehydrogenase (PDH) plays a major role in the metabolic fate of pyruvate (oxidation vs. lactate production), we assessed the changes in its activity and regulation. Here we report that when run under hypoxia, 24H mice exhibited the highest blood and intramuscular lactate of all groups, while the 1WH group approached N group values. Concomitantly, the 24H group exhibited the lowest PDH activity, associated with a higher phosphorylation (inactive) state of the Ser(232) residue of PDH, a site specific to PDH kinase-1 (PDK1). Furthermore, protein levels of PDK1 and its regulator, the hypoxia inducible factor-1α (HIF-1α), were both elevated in the 24H group compared with N and 1WH groups. Overall, our results point to a novel mechanism in muscle where the HIF-1α pathway is desensitized in the transition from acute to chronic hypoxia, leading to a reestablishment of PDH activity and a reduction in lactate production by the exercising muscles.

Genome duplication events have led to a diversification in the CPT I gene family in fish.

The enzyme carnitine palmitoyltransferase (CPT) I is a major regulator of mitochondrial fatty acid oxidation in vertebrates. Numerous genome duplication events throughout evolution have given rise to three (in mammals) or multiple (in fish) genetically and functionally different isoforms of this enzyme. In particular, these isoforms represent a diversification of kinetic and regulatory properties stemming from mutations at the genomic and proteomic levels. Phylogenetic reconstructions reveal a comprehensive view of the CPT I family in vertebrates and genomic modifications leading to structural changes in proteins and functional differences between tissues and taxa. In a model fish species (rainbow trout), the presence of five CPT I isoforms suggests repeated duplication events in bony fishes and salmonids. Subsequently, an array of nucleotide and amino acid substitutions in the isoforms may contribute to a tissue-specific and a previously observed species-specific difference in the IC(50) for malonyl-CoA. Moreover, all five isoforms are expressed in trout at the mRNA level in skeletal muscle, heart, liver, kidney, and intestine. In general, transcript levels of the beta-isoforms were higher in muscle tissues, while levels of the alpha-isoforms were higher in other tissues. Rainbow trout also exhibit developmental plasticity in relative mRNA expression of CPT I isoforms from fry to juvenile to adult stage. Thus the evolution of CPT I has resulted in a very diverse family of isoforms. These differences represent a degree of specificity in the ability of species to regulate function at the protein and tissue levels, which, in turn, may allow for precise control of lipid oxidation in individual tissues during physiological perturbations.

Effects of dietary fatty acid composition on the regulation of carnitine palmitoyltransferase (CPT) I in rainbow trout (Oncorhynchus mykiss).

Dietary fatty acid composition, particularly polyunsaturated fatty acids, can affect both genetic and non-genetic regulatory mechanisms of carnitine palmitoyltransferase (CPT) I, the main regulatory enzyme of mitochondrial fatty acid oxidation. We aimed to determine how these regulatory mechanisms were affected by changes in the fatty acid composition of the diet in fish. Specifically, we fed rainbow trout (Oncorhynchus mykiss) either a high polyunsaturated fatty acid (PUFA) diet, a high saturated fatty acid (SFA) diet or a mixed fatty acid control (CTL) diet for 8 weeks to determine if modifications of the dietary fatty acids would affect 1) the genetic expression of CPT I and its transcription factor peroxisome proliferator activated receptor (PPAR), 2) the mitochondrial membrane composition and if these modifications would affect CPT I sensitivity to malonyl-CoA, and 3) levels of malonyl-CoA in the tissues. We found that fish fed the high PUFA diet significantly increased CPT I mRNA expression in red muscle, liver and adipose tissue, while PPAR alpha and beta expressions were variable across tissues. Few significant changes were observed in the mitochondrial membrane composition with the exception of DHA in the red muscle. There were no significant differences in CPT I sensitivity to malonyl-CoA or the malonyl-CoA content of the tissues with either experimental diet. Our present data suggest that changes in gene expression of CPT I and PPARs is the main regulatory mechanism controlling CPT I function in fish using our experimental diet.

Intertissue regulation of carnitine palmitoyltransferase I (CPTI): mitochondrial membrane properties and gene expression in rainbow trout (Oncorhynchus mykiss).

Carnitine palmitoyltransferase (CPT) I is regulated by several genetic and non-genetic factors including allosteric inhibition, mitochondrial membrane composition and/or fluidity and transcriptional regulation of enzyme content. To determine the intrinsic differences in these regulating factors that may result in differences between tissues in fatty acid oxidation ability, mitochondria were isolated from red, white and heart muscles and liver tissue from rainbow trout. Maximal activity (V(max)) for beta-oxidation enzymes and citrate synthase per mg tissue protein as well as CPT I in isolated mitochondria followed a pattern across tissues of red muscle>heart>white muscle>liver suggesting both quantitative and qualitative differences in mitochondria. CPT I inhibition showed a similar pattern with the highest malonyl-CoA concentration to inhibit activity by 50% (IC(50)) found in red muscle while liver had the lowest. Tissue malonyl-CoA content was highest in white muscle with no differences between the other tissues. Interestingly, the gene expression profiles did not follow the same pattern as the tissue enzyme activity. CPT I mRNA expression was greatest in heart>red muscle>white muscle>liver. In contrast, PPARalpha mRNA was greatest in the liver>red muscle>heart>white muscle. There were no significant differences in the mRNA expression of PPARbeta between tissues. As well, no significant differences were found in the mitochondrial membrane composition between tissues, however, there was a tendency for red muscle to exhibit higher proportions of PUFAs as well as a decreased PC:PE ratio, both of which would indicate increased membrane fluidity. In fact, there were significant correlations between IC(50) of CPT I for malonyl-CoA and indicators of membrane fluidity across tissues. This supports the notion that sensitivity of CPT I to its allosteric regulator could be modulated by changes in mitochondrial membrane composition and/or fluidity.